Construction And Characterization Of A Dual-variable-domain Neutralizing Antibody Against Dengue Virus With High Affinity

Dengue Viruses (DENV) belong to Flaviviruses, which also include West Nile(WNV), Japanese encephalitis (JEV), and tick-borne encephalitis viruses (TBEV). Thegenome of DENV is plus-strand RNA packed by envelop, which code three structureproteins (C, prM/M, E) and seven non-structure proteins. The E protein is the majorglycoprotein on the surface of virions, it supply main immunogenicity. According to thedifference of immunogenicity of the E protein, DENV were divided into four serotypes(DENV1-4). Clinically, DENV is the agent of dengue fever (DF), dengue hemorrhagicfever (DHF) and dengue shock syndrome (DSS). Over50million people get infected allover the world each year, and tens of thousands people dead finally. However, there is nothaving an effective treatment method for DENV infection but supportive treatment. Sodeveloping a novel drug that can prevent and therapy DENV infection effectively is gettingincreasingly important.With the development of antibody technology, using neutralizing antibody to preventand treatment virus infection was receiving more and more attention. Compare to thetraditional drugs, antibody treatments have the advantages of specificity and efficiency.Attachment and fusion are two key processes of dengue virus infection. When the virusattach to cell surface, EDⅢ first bind to the receptor on the cytomembrane. Then virionswere swallowed into the cell by receptor-mediated endocytosis. DC-SIGN(dendritic-cell-specific ICAM-grabbing non-integrin), GRP78/Bip (glucose-regulatingprotein78), and CD14-associated molecules have been suggested as primary receptors fordengue virus. So most of epitopes that recognized by neutralizing antibodies located at thisfunction domain. This kind of antibodies prevents infection through inhibit virus binding tothe receptor. After dengue viruses entered cells, they first packaged by the endosome. Onlyafter envelop of the virus fuse with endosome, do RNA of the virus will be released intocells. The fusion loop at the top of EDⅡ mediate this process. And the amino acidsequences of the fusion loop were highly conservative in flavivirus. Therefore, most ofcross-reactive neutralizing antibodies recognize epitopes on this region. Up to now, therewere several antibodies that could neutralize dengue virus. The mAb1A1D and2A10havethe ability of neutralizing multi-serotype of DENV,1A1D inhibit attachment of the virusby binding with EDⅢ,2A10inhibit fusion of the virus by binding EDⅡ. If we combinetwo kinds of antibodies, we will get better neutralizing effect. But we also have to solve many problems about safe and cost.Wu et al. described a new approach for producing bispecific tetravalentimmunoglobulin G (IgG)-like molecules, which they named dual-variable domainimmunoglobulin (DVD-Ig). It supply us a new method that we can construct two differentvariable domains on one antibody molecule and keep the original affinity and neutralizingability of them. Consequently, it can avoid troubles that taken by multi-using of severalantibodies, and it is more safety and more valuable for clinical use. Furthermore, we couldeliminate the ADE effect by introducing Fc mutations which could abrogate the binding ofFc and Fc receptors.In this study, we intended to construct a novel DVD antibody, which could recognizetwo different epitopes. And it has higher affinity to improve the neutralizing ability. Finally,we eliminate the ADE effect by introducing the deletion mutation of the Fc domain.Firstly, we link variable regions of1A1D and2A10by nine amino acid linker. Thenwe constructed the light chain and the heavy chain of the DVD antibody. Both of themwere inserting into the expression vector. Then, CHO cells were co-transfected with heavychain and light chain expression vectors using lipofectamine2000reagent. The cell cloneswith higher expression levels were selected and expanded in serum-free medium. Theculture medium were collected and purified by proteinA affinity column to get theDVD-Ig.Secondly, we test binding activities of two DVD antibodies with different orientationof variable regions by western blot and ELISA. The binding activity of DVD-1A1D-2A10is better than DVD-2A10-1A1D. The DVD-1A1D-2A10was confirmed that could bindwith virus normally by IFA, and binding curves with EDⅠ/Ⅱ and EDⅢ reflect that thetetravalent structure of DVD-Ig did not affect binding ability of it, in addition, compare todivalent antibodies, it has better affinity with a whole E protein. The neutralizing ability ofDVD-Ig was determined by a standard plaque reduction neutralization test (PRNT) in vitro.The results indicated that the DVD-Ig has more potent neutralizing ability than mAbs. Tofurther confirm the function of the DVD-1A1D-2A10, we used suckling mice model to testthe neutralizing ability in vivo, and tested the preventive and therapeutic effect by injecting50μg antibodies before or after infection.Finally, we eliminated the ADE effect of the DVD-Ig by introducing Fc deletionmutations, and confirmed that did not affect the neutralizing ability. This shown that wesuccessfully construct a high affinity dual-variable-domain neutralizing antibody against multi-serotype of DENV.